Carbohydrate–PNA and Aptamer–PNA Conjugates for the Spatial Screening of Lectins and Lectin Assemblies

Nucleic acid architectures offer intriguing opportunities for the interrogation of structural properties of protein receptors. In this study, we performed a DNA‐programmed spatial screening to characterize two functionally distinct receptor systems: 1) structurally well‐defined Ricinus communis agglutinin (RCA₁₂₀), and 2) rather ill‐defined assemblies of L‐selectin on nanoparticles and leukocytes. A robust synthesis route that allowed the attachment both of carbohydrate ligands—such as N‐acetyllactosamine (LacNAc), sialyl‐Lewis‐X (sLe^X), and mannose—and of a DNA aptamer to PNAs was developed. A systematically assembled series of different PNA–DNA complexes served as multivalent scaffolds to control the spatial alignments of appended lectin ligands. The spatial screening of the binding sites of RCA₁₂₀ was in agreement with the crystal structure analysis. The study revealed that two appropriately presented LacNAc ligands suffice to provide unprecedented RCA₁₂₀ affinity (K_D=4 μM ). In addition, a potential secondary binding site was identified. Less dramatic binding enhancements were obtained when the more flexible L‐selectin assemblies were probed. This study involved the bivalent display both of the weak‐affinity sLe^X ligand and of a high‐affinity DNA aptamer. Bivalent presentation led to rather modest (sixfold or less) enhancements of binding when the self‐assemblies were targeted against L‐selectin on gold nanoparticles. Spatial screening of L‐selectin on the surfaces of leukocytes showed higher affinity enhancements (25‐fold). This and the distance–activity relationships indicated that leukocytes permit dense clustering of L‐selectin.     

High‐Affinity Carbohydrate Binding by Trimeric Benzoboroxoles Measured on Carbohydrate Arrays

Carbohydrates are involved in a wide range of biological processes of pharmaceutical relevance. The selective recognition of carbohydrates is therefore of great interest in biology and medicine. In this study we present the synthesis of fluorescent multimeric benzoboroxoles and the analysis of multivalent binding processes to immobilized carbohydrate arrays by fluorescence spectroscopy. We observed high binding affinities of trimeric benzoboroxoles by determination of K_Dsurf values for their interaction with α‐Gal on glass chips. The observed K_Dsurf values were in the mid‐nM range (49 and 104 nM ) and are comparable to the K_Dsurf values for binding of natural lectins, such as that of ConA to immobilized α‐Man (79 nM ). The array technology was found to be an excellent tool for studying the binding processes of multivalent lectin mimetics with respect to profiling and quantitation.     

Assessing Carbohydrate–Carbohydrate Interactions by NMR Spectroscopy: The Trisaccharide Epitope from the Marine Sponge Microciona prolifera

Weak recognition processes : Weak calcium‐mediated carbohydrate–carbohydrate interactions have been detected by DOSY and TRNOESY NMR methods by employing a gold glyconanoparticle as a multivalent system. In addition, 3D models of trisaccharide‐Ca^II‐trisaccharide complexes based on results from molecular dynamics simulations are proposed.

     

Human Macrophage Galactose-Type Lectin (MGL) Recognizes the Outer Core of Escherichia coli Lipooligosaccharide

Carbohydrate–lectin interactions intervene in and mediate most biological processes, including a crucial modulation of immune responses to pathogens. Despite growing interest in investigating the association between host receptor lectins and exogenous glycan ligands, the molecular mechanisms underlying bacterial recognition by human lectins are still not fully understood. Herein, a novel molecular interaction between the human macrophage galactose-type lectin (MGL) and the lipooligosaccharide (LOS) of Escherichia coli strain R1 is described. Saturation transfer difference NMR spectroscopy analysis, supported by computational studies, demonstrated that MGL bound to the purified deacylated LOS_R1 mainly through recognition of its outer core and established crucial interactions with the terminal Galα(1,2)Gal epitope. These results assess the ability of MGL to recognise glycan moieties exposed on Gram-negative bacterial surfaces.     

A Novel Chemoenzymatic Synthesis of Sulfated Type 2 Tumor‐Associated Carbohydrate Antigens by Transglycosylation of Sulfated Lewis X Oxazoline Catalyzed by Keratanase II

Sulfated type 2 carbohydrate chains are known tumor‐associated carbohydrate antigens (TACAs). Many reports on cancer vaccines employing TACAs as specific antigens have been published, but structurally specified sulfated TACAs have not been used because of the low natural abundance and difficulties in chemical synthesis. We demonstrate for the first time the synthesis of the sulfated type 2 TACAs with an l ‐fucose branch by keratanase‐II‐catalyzed transglycosylation of the sulfated Lewis X (Galβ(1→4)[Fucα(1→3)]GlcNAc(6‐OSO₃^?); su‐Le^x) oxazoline derivative. Two keratanase IIs (from Bacillus sp. Ks36 and Bacillus circulans KsT202) efficiently catalyzed the transglycosylation reaction of the su‐Le^x oxazoline derivative, thereby giving the su‐Le^x dimer as the main product in good yields. Structural analysis of the oligomers confirmed exclusive formation of the β(1→3) glycosidic bond.     

Unraveling the Interaction between the LPS O‐Antigen of Burkholderia anthina and the 5D8 Monoclonal Antibody by Using a Multidisciplinary Chemical Approach,with Synthesis,NMR, and Molecular Modeling Methods

The interaction between the O‐chain from the lipopolysaccharide from Burkholderia anthina and a lipopolysaccharide‐specific monoclonal antibody (5D8) has been studied at high resolution by NMR spectroscopy. In particular, the 5D8‐bound epitope of the saccharide entity has been unraveled by a combination of saturation transfer difference (STD) and transferred NOESY (tr‐NOESY) experiments performed on the 5D8/polysaccharide complex. To dissect the fine details of the molecular recognition events, further experiments with simpler carbohydrate ligands were carried out. Thus, experiments were also performed with ad hoc synthesized trisaccharide and hexasaccharide O‐antigen repeating units. By using this multidisciplinary approach (chemical synthesis, NMR spectroscopy and molecular dynamics simulation), determination of the binding epitope and the contribution to the binding of the sugar units composing the O‐chain have been determined.     

Human Milk Oligosaccharide 2′-Fucosyllactose Inhibits Ligand Binding to C-Type Lectin DC-SIGN but Not to Langerin

Human milk oligosaccharides (HMOs) and their most abundant component, 2′-Fucosyllactose (2′-FL), are known to be immunomodulatory. Previously, it was shown that HMOs and 2′-FL bind to the C-type lectin receptor DC-SIGN. Here we show, using a ligand-receptor competition assay, that a whole mixture of HMOs from pooled human milk (HMOS) and 2′-FL inhibit the binding of the carbohydrate-binding receptor DC-SIGN to its prototypical ligands, fucose and the oligosaccharide Lewis-B, (Le^b) in a dose-dependent way. Interestingly, such inhibition by HMOS and 2′-FL was not detected for another C-type lectin, langerin, which is evolutionarily similar to DC-SIGN. The cell-ligand competition assay using DC-SIGN expressing cells confirmed that 2′-FL inhibits the binding of DC-SIGN to Le^b. Molecular dynamic (MD) simulations show that 2′-FL exists in a preorganized bioactive conformation before binding to DC-SIGN and this conformation is retained after binding to DC-SIGN. Le^b has more flexible conformations and utilizes two binding modes, which operate one at a time via its two fucoses to bind to DC-SIGN. Our hypothesis is that 2′-FL may have a reduced entropic penalty due to its preorganized state, compared to Le^b, and it has a lower binding enthalpy, suggesting a better binding to DC-SIGN. Thus, due to the better binding to DC-SIGN, 2′-FL may replace Le^b from its binding pocket in DC-SIGN. The MD simulations also showed that 2′-FL does not bind to langerin. Our studies confirm 2′-FL as a specific ligand for DC-SIGN and suggest that 2′-FL can replace other DC-SIGN ligands from its binding pocket during the ligand-receptor interactions in possible immunomodulatory processes.     

Carbohydrate-mediated targeting of antigen to dendritic cells leads to enhanced presentation of antigen to T cells

The unique therapeutic value of dendritic cells (DCs) for the treatment of allergy, autoimmunity and transplant rejection is predicated upon our ability to selectively deliver antigens, drugs or nucleic acids to DCs in vivo. Here we describe a method for delivering whole protein antigens to DCs based on carbohydrate-mediated targeting of DC-expressed lectins. A series of synthetic carbohydrates was chemically-coupled to a model antigen, ovalbumin (OVA), and each conjugate was evaluated for its ability to increase the efficiency of antigen presentation by murine DCs to OVA-specific T cells (CD4(+) and CD8(+)). In vitro data are presented that demonstrate that carbohydrate modification of OVA leads to a 50-fold enhancement of presentation of antigenic peptide to CD4(+) T cells. A tenfold enhancement is observed for CD8(+) T cells; this indicates that the targeted lectin(s) can mediate cross-presentation of antigens on MHC class I. Our data indicate that the observed enhancements in antigen presentation are unique to OVA that is conjugated to complex oligosaccharides, such as a high-mannose nonasaccharide, but not to monosaccharides. Taken together, our data suggest that a DC targeting strategy that is based upon carbohydrate-lectin interactions is a promising approach for enhancing antigen presentation via class I and class II molecules.     

Lactose‐Functionalized Dendrimers Arbitrate the Interaction of Galectin‐3/MUC1 Mediated Cancer Cellular Aggregation

By using lactose‐functionalized poly(amidoamine) dendrimers as a tunable multivalent platform, we studied cancer cell aggregation in three different cell lines (A549, DU‐145, and HT‐1080) with galectin‐3. We found that small lactose‐functionalized G(2)‐dendrimer 1 inhibited galectin‐3‐induced aggregation of the cancer cells. In contrast, dendrimer 4 (a larger, generation 6 dendrimer with 100 carbohydrate end groups) caused cancer cells to aggregate through a galectin‐3 pathway. This study indicates that inhibition of cellular aggregation occurred because 1 provided competitive binding sites for galectin‐3 (compared to its putative cancer cell ligand, TF‐antigen on MUC1). Dendrimer 4 , in contrast, provided an excess of ligands for galectin‐3 binding; this caused crosslinking and aggregation of cells to be increased.     

Rapid Screening of Lectins for Multivalency Effects with a Glycodendrimer Microarray

Multivalency is an important phenomenon in protein–carbohydrate interactions. In order to evaluate glycodendrimers as multivalent inhibitors of carbohydrate binding proteins, we displayed them on a microarray surface. Valencies were varied from 1 to 8, and corrections were made for the valencies so that all surfaces contained the same amount of the sugar ligand. Five different carbohydrates were attached to the dendrimers. A series of fluorescent lectins was evaluated, and for each of them a binding profile was obtained from a single experiment showing both the specificity of the lectin for a certain sugar and whether it prefers multivalent ligands or not. Very distinct binding patterns were seen for the various lectins. The results were rationalized with respect to the interbinding distances of the lectins.     

Multimeric Lactoside “Click Clusters” as Tools to Investigate the Effect of Linker Length in Specific Interactions with Peanut Lectin,Galectin‐1, and ‐3

Multimeric lactosides based on carbohydrate scaffolds with valencies ranging from 1 to 4 and different linker lengths were synthesized by a copper‐catalyzed azide–alkyne cycloaddition (CuAAC). The binding affinities and crosslinking abilities of the new “click clusters” toward biologically relevant galectins (gal‐1, gal‐3) and peanut lectin were evaluated by fluorescent polarization assay (FPA) and enzyme‐linked lectin assay (ELLA), respectively. FPA indicated that the binding affinities of the synthetic multilactosides towards the galectins increased proportionally with their lactosyl content, without significant differences due to the spacer length. ELLA evidenced a modest cluster effect for the multivalent conjugates, with a relative potency per lactoside ranging from 2.1 to 3.2. Nearly identical binding affinities were recorded for derivatives differing in the length of the linkers, in agreement with the FPA data. These results demonstrate that this parameter does not significantly influence the recognition process when interactions occur at a single lectin site. Molecular dynamics revealed that glycoconjugates adopt a pseudoglobular structure with a random localization of the lactoside residues. These spatial distributions were observed irrespective of the linker length; this explains the virtually equal affinities recorded by ELLA. In contrast, two‐site “sandwich” ELLA clearly revealed that multivalent derivatives bearing the longest spacers were more efficient for crosslinking lectins. Intrinsic affinities, devoid of aggregation effects, and crosslinking capabilities are, therefore, not directly related phenomena that must be taking into consideration in neoglycoconjugate design for specific applications.     

Cross-Linking Effects Dictate the Preference of Galectins to Bind LacNAc-Decorated HPMA Copolymers

The interaction of multi-LacNAc (Galβ1-4GlcNAc)-containing N-(2-hydroxypropyl) methacrylamide (HPMA) copolymers with human galectin-1 (Gal-1) and the carbohydrate recognition domain (CRD) of human galectin-3 (Gal-3) was analyzed using NMR methods in addition to cryo-electron-microscopy and dynamic light scattering (DLS) experiments. The interaction with individual LacNAc-containing components of the polymer was studied for comparison purposes. For Gal-3 CRD, the NMR data suggest a canonical interaction of the individual small-molecule bi- and trivalent ligands with the lectin binding site and better affinity for the trivalent arrangement due to statistical effects. For the glycopolymers, the interaction was stronger, although no evidence for forming a large supramolecule was obtained. In contrast, for Gal-1, the results indicate the formation of large cross-linked supramolecules in the presence of multivalent LacNAc entities for both the individual building blocks and the polymers. Interestingly, the bivalent and trivalent presentation of LacNAc in the polymer did not produce such an increase, indicating that the multivalency provided by the polymer is sufficient for triggering an efficient binding between the glycopolymer and Gal-1. This hypothesis was further demonstrated by electron microscopy and DLS methods.     

Carbohydrate-based Biomedical Copolymers for Targeted Delivery of Anticancer Drugs

A variety of strategies and carrier molecules have been used to direct therapeutic agents to tumor sites. The incorporation of a specific targeting moiety to drug carrier may result in active drug uptake by malignant cells. Carbohydrates are important mediators of cell–cell recognition events and have been implicated in related processes such as cell signaling regulation, cellular differentiation, and immune response. The biocompatibility of carbohydrates and their ability to be specifically recognized by cell-surface receptors indicate their potential utility as ligands in targeted drug delivery for therapeutic applications. Yet, carbohydrates are not ideal targeting ligands because they are difficult to synthesize, bind weakly to carbohydrate receptors, and are prone to suffer from enzyme degradation due to labile glycosidic linkages. This review describes the design and development of HPMA-based biomedical copolymers to facilitate the selective delivery of drugs to tumor tissues via carbohydrate–endogenous lectin interactions. Various carbohydrate-decorated HPMA copolymer–drug conjugates are presented and the application of the copolymers for drug delivery is discussed. Current efforts to increase the affinity of carbohydrate ligands for their target receptors through multivalent display are also discussed. These novel HPMA copolymer carbohydrate conjugates hold promise as clinically relevant drug delivery systems for cancer therapy.     

Exploring the Structural Space of the Galectin‐1–Ligand Interaction

Galectin‐1 is a tumor‐associated protein recognizing the Galβ1‐4GlcNAc motif of cell‐surface glycoconjugates. Herein, we report the stepwise expansion of a multifunctional natural scaffold based on N‐acetyllactosamine (LacNAc). We obtained a LacNAc mimetic equipped with an alkynyl function on the 3′‐hydroxy group of the disaccharide facing towards a binding pocket adjacent to the carbohydrate‐recognition domain. It served as an anchor motif for further expansion by the Sharpless–Huisgen–Meldal reaction, which resulted in ligands with a binding mode almost identical to that of the natural carbohydrate template. X‐ray crystallography provided a structural understanding of the galectin‐1–ligand interactions. The results of this study enable the development of bespoke ligands for members of the galectin target family.     

Synthesis and Characterization of Oriented Glyco‐Capturing Macroligand

An oriented glyco‐capturing macroligand was synthesized by site‐specific immobilization of an O‐cyanate chain‐end‐functionalized boronic acid containing polymer (boropolymer) onto an amine surface. The O‐cyanate chain‐end‐functionalized boropolymer was synthesized by arylamine‐initiated cyanoxyl‐mediated free‐radical polymerization in a one‐pot fashion. The chain‐end O‐cyanate was confirmed by ¹³C NMR spectroscopy. The specific carbohydrate‐binding capacity of the boropolymer was evaluated by an alizarin red S assay. Oriented and covalent immobilization of the O‐cyanate chain‐end‐functionalized boropolymer onto the amine‐modified solid surfaces and its specific glyco‐capturing capacity were confirmed by the quartz crystal microbalance (QCM) and atomic force microscopy (AFM) techniques. The oriented multivalent glyco‐capturing ligand can be used for efficient carbohydrate and glycoconjugate purification and identification, and thus is expected to constitute a core strategy of glycomics and glycoproteomics and carbohydrate‐sensing applications.     

Determination of Carbohydrate‐Binding Preferences of Human Galectins with Carbohydrate Microarrays

Galectins are a class of carbohydrate‐binding proteins named for their galactose‐binding preference and are involved in a host of processes ranging from homeostasis of organisms to immune responses. As a first step towards correlating the carbohydrate‐binding preferences of the different galectins with their biological functions, we determined carbohydrate recognition fine‐specificities of galectins with the aid of carbohydrate microarrays. A focused set of oligosaccharides considered relevant to galectins was prepared by chemical synthesis. Structure–activity relationships for galectin–sugar interactions were determined, and these helped in the establishment of redundant and specific galectin actions by comparison of binding preferences. Distinct glycosylations on the basic lactosyl motifs proved to be key to galectin binding regulation—and therefore galectin action—as either high‐affinity ligands are produced or binding is blocked. High‐affinity ligands such as the blood group antigens that presumably mediate particular functions were identified.     

Vector and Host C-Type Lectin Receptor (CLR)–Fc Fusion Proteins as a Cross-Species Comparative Approach to Screen for CLR–Rift Valley Fever Virus Interactions

Rift Valley fever virus (RVFV) is a mosquito-borne bunyavirus endemic to Africa and the Arabian Peninsula, which causes diseases in humans and livestock. C-type lectin receptors (CLRs) represent a superfamily of pattern recognition receptors that were reported to interact with diverse viruses and contribute to antiviral immune responses but may also act as attachment factors or entry receptors in diverse species. Human DC-SIGN and L-SIGN are known to interact with RVFV and to facilitate viral host cell entry, but the roles of further host and vector CLRs are still unknown. In this study, we present a CLR–Fc fusion protein library to screen RVFV–CLR interaction in a cross-species approach and identified novel murine, ovine, and Aedes aegypti RVFV candidate receptors. Furthermore, cross-species CLR binding studies enabled observations of the differences and similarities in binding preferences of RVFV between mammalian CLR homologues, as well as more distant vector/host CLRs.     

Self-Assembling Lectin Nano-Block Oligomers Enhance Binding Avidity to Glycans

Lectins, carbohydrate-binding proteins, are attractive biomolecules for medical and biotechnological applications. Many lectins have multiple carbohydrate recognition domains (CRDs) and strongly bind to specific glycans through multivalent binding effect. In our previous study, protein nano-building blocks (PN-blocks) were developed to construct self-assembling supramolecular nanostructures by linking two oligomeric proteins. A PN-block, WA20-foldon, constructed by fusing a dimeric four-helix bundle de novo protein WA20 to a trimeric foldon domain of T4 phage fibritin, self-assembled into several types of polyhedral nanoarchitectures in multiples of 6-mer. Another PN-block, the extender PN-block (ePN-block), constructed by tandemly joining two copies of WA20, self-assembled into cyclized and extended chain-type nanostructures. This study developed novel functional protein nano-building blocks (lectin nano-blocks) by fusing WA20 to a dimeric lectin, Agrocybe cylindracea galectin (ACG). The lectin nano-blocks self-assembled into various oligomers in multiples of 2-mer (dimer, tetramer, hexamer, octamer, etc.). The mass fractions of each oligomer were changed by the length of the linkers between WA20 and ACG. The binding avidity of the lectin nano-block oligomers to glycans was significantly increased through multivalent effects compared with that of the original ACG dimer. Lectin nano-blocks with high avidity will be useful for various applications, such as specific cell labeling.     

Copolymerization of potassium chloroacetate and potassium N‐chloroacetyl‐6‐aminohexanoate

The polymerization kinetics of potassium chloroacetate (MGL), potassium N‐chloroacetyl‐6‐aminohexanoate (MEA), and their mixtures was studied by Fourier transform infrared spectroscopy. The bulk polycondensation reaction was faster for MEA than for MGL but kinetic differences in the selected temperature range (110–130°C) were not large enough to make unfeasible copolymerization of both monomers. A decrease in the activation energy was deduced for the polycondensation of monomer mixtures with respect to that determined for the homopolymerization reaction of the predominant neat monomer. differential scanning calorimetry data also showed significant differences in the exothermic polycondensation peaks that suggested an effective copolymerization reaction and favored the kinetic process over the corresponding homopolymerization. The resulting new poly(ester amide)s were characterized by spectroscopy and thermal analysis. ¹H NMR spectra of samples with high MEA content revealed the existence of hetero‐sequences whose ratio was slightly lower than that expected for a random polymerization of the two monomers. Samples with high molecular weights were only attained when the MGL molar ratio in the monomer mixture was lower than 65%. Calorimetric data showed that all samples were thermally stable and became amorphous for intermediate compositions. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012